Application Note

Ultra-Sensitive Quantification Of Genome Editing Events Using Droplet Digital™ PCR

Source: Bio-Rad

By Jennifer R. Berman, Samantha Cooper, Bin Zhang, George Karlin-Neumann, Claudia Litterst, Yann Jouvenot, Eli Hefner, Yuichiro Miyaoka, and Bruce R. Conklin

Genomic Laboratory Services

Genome editing tools including TALENs and the CRISPR/Cas9 system have revolutionized our ability to edit the genome of any cell, including human induced pluripotent stem cells (iPSCs). Sequence-specific nucleases induce double-strand breaks or nicks at target sites, activating the DNA repair pathways of non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ produces small insertions or deletions (indels) and is useful for disrupting gene function. HDR can induce precise gene repair of one to thousands of base pairs in the presence of a homologous donor molecule, allowing for correction of point mutations and introduction of exogenous sequences. Genome editing is an increasingly common part of the molecular biologist’s toolkit and is being actively developed for therapeutic indications.

While powerful, the efficiency of gene editing is cell type–dependent and often low (<5%), particularly in primary cells or iPSCs. Sanger sequencing and gel-based methods lack the sensitivity required to detect such events in non-clonal cell populations. Detection by next-generation sequencing (NGS) presents hurdles in terms of cost and workflow, and NGS sample prep can introduce bias for or against edited alleles.

Droplet Digital PCR (ddPCR™) enables ultra-sensitive absolute quantification of genome editing events. Here we introduce ddPCR assay strategies for the detection of HDR- and NHEJedited alleles. Using these assays we can detect alleles in edited samples present at frequencies of less than 0.5%. These methods are useful for ultra-sensitive detection of edited alleles and offer a rapid, low-cost readout for technical optimization of genome editing protocols.

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